DyeEx 96 Kit

For removal of unincorporated dye terminators from 1–96 sequencing reactions

Fast procedure with only two short centrifugation steps

Ready-to-use prehydrated gel-filtration material

Efficient removal of any dye terminator

DyeEx Kits provide either spin columns or 96-well plates and use gel-filtration technology to remove dye terminators from sequencing reactions. Sequencing reactions are loaded onto the pre-hydrated gel-filtration material. After a short centrifugation step, the reactions are ready to be loaded onto a capillary sequencer. Unincorporated dye terminators are retained in the gel matrix.

DyeEx Kits remove any type of dye terminator from 10–20 µl sequencing reactions including BigDye, dRhodamine dye, Rhodamine, DYEnamic ET, and WellRED terminators. After cleanup, the sequencing reactions can be separated on any capillary sequencer. We recommend the DyeEx 96 Kit using the optimized protocol. Signal intensities are high, resulting in long read lengths (see figure "Long-read sequence").

High-quality sequencing

DyeEx Kits are optimized for fast and convenient dye-terminator removal leading to high-quality sequencing results. In contrast, sequencing-reaction cleanup by ethanol precipitation is very time consuming (see table "Comparison of DyeEx procedure and ethanol precipitation") and inefficient. The failure to remove dye terminators efficiently often leads to the appearance of dye blobs in sequencing data making stretches of sequence unreadable. Using DyeEx Kits for sequencing reaction cleanup ensures that the reactions loaded onto sequencing instruments are free of dye terminators. Without dye blobs, the sequence is easily readable throughout (see figure "Superior sequencing results").

N.A.: not applicable
* Does not include time required to dry down samples before gel loading (where applicable).† Using the standard protocol.

Principle

The DyeEx procedure uses gel filtration to quickly and efficiently remove unincorporated terminators from sequencing reactions. Removal of dye terminators is important to prevent the unincorporated dye terminators from interfering with analysis of sequencing results. The DyeEx gel-filtration material consists of spheres with uniform pores and separates molecules according to molecular weight. When sequencing reaction mixtures are applied to DyeEx columns, dye terminators diffuse into the pores and are retained in the gel-filtration material, while labeled DNA fragments are excluded and recovered in the flow-through (see figure "DyeEx separation principle").

Procedure

The DyeEx 96 Kit is used with a suitable table-top centrifuge equipped with a rotor with swing-out buckets, such as the QIAGEN 96-Well-Plate Centrifugation System. Dye-terminator removal with DyeEx Kits is fast because the procedure is so simple (see flowchart "DyeEx 96 procedure"). A quick centrifugation step removes storage buffer from the columns, the sequencing samples are loaded, and a second centrifugation step removes unincorporated dye terminators. Samples are then ready for direct loading onto a capillary sequencer or can be dried, redissolved, and loaded onto a sequencing gel.

Applications

DyeEx Kits remove any type of dye terminator from 10–20 µl sequencing reactions including BigDye, dRhodamine dye, Rhodamine, DYEnamic ET, and WellRED terminators. After cleanup, the sequencing reactions can be separated on any capillary sequencer. Signal intensities are high, resulting in long read lengths.